Abstract
Breast cancer (BC) is now the most frequently diagnosed malignancy and primary cause of cancer-related death among women worldwide, including in New Zealand. Metastasis, the spread of cancer cells from primary breast tumours to lymph nodes and distant sites, remains the main driver of breast cancer mortality, accounting for nearly 90% of deaths. The rising rates of breast cancer mortalities and the lack of effective treatments for patients with metastasis highlight the urgent need to uncover novel molecular targets for improved clinical diagnosis and therapeutic interventions. The epithelial sodium channel (ENaC), a trimer composed of α- (or δ-), β-, and γ-subunits that facilitates sodium ion reabsorption, has emerged as a significant player in the tumorigenesis and metastatic progression of various cancers, including breast cancer. Previous research in the McDonald laboratory revealed that increased expression of the α-subunit of ENaC significantly reduces breast cancer cell proliferation and migration. Bioinformatic analyzes of breast cancer patients further suggest a link between increased α-ENaC expression and reduced epithelial-mesenchymal plasticity (EMP), a process known to drive metastasis by enabling cells to transition between epithelial and more migratory and invasive mesenchymal cell states. However, the role of α-ENaC in EMP and other metastasis-associated processes and cellular changes has not been comprehensively investigated. This PhD thesis has, therefore, set out to investigate the impacts of α-ENaC modulation on cell invasion, EMP and the biomechanical properties of breast cancer cells.
To investigate the role of α-ENaC in EMP, the effects of transiently overexpressing or knocking down α-ENaC on the mRNA and protein levels of classical EMP markers typically used to characterize cell phenotypes (E-cadherin, N-cadherin, Vimentin, Twist and Snail) were assessed and compared across five phenotypically different human breast cell lines (epithelial-like MCF-7 and T-47D; mesenchymal-like BT-549 and MDA-MB-231; non-tumorigenic MCF-10A). It was hypothesized that an increase in α-ENaC levels would upregulate the epithelial marker, E-cadherin and downregulate the mesenchymal markers, and vice versa for reduced α-ENaC levels. Contrarily, expression analysis using RT-qPCR and western blot revealed that these EMP markers were variably altered with the modulation of α-ENaC expression in a cell-type-dependent manner.
Given the concerns of inconsistent alteration of α-ENaC expression with the transient transfection method, a stable α-ENaC-overexpressing MDA-MB-231 triple-negative breast cancer (TNBC) cell line, previously established in the McDonald laboratory, was employed to further elucidate α-ENaC’s role in breast cancer metastasis. This cell line has been shown to exhibit reduced migration and proliferation. Herein, using a Matrigel transwell invasion assay, it was further discovered that stably overexpressing α-ENaC suppressed the invasive ability of MDA-MB-231 cells (N=4, p<0.01). In addition, there were no significant changes observed in the expression of EMP markers (N=6, p>0.05). Conversely, stable α-ENaC overexpression in MDA-MB-231 cells resulted in the acquisition of epithelial-like morphology (N=4, p<0.001), increased F-actin density (N=4, p<0.0001), disorganized F-actin network, and reduced localization of the invadopodia protein, cortactin to the cell edges (N=4, p=0.057). Altogether, these findings suggest that EMP is not the primary mechanism underlying α-ENaC’s role in breast cancer cells; instead, an F-actin network/cortactin-dependent mechanism that modulates cell morphology and, potentially, the formation of cell protrusions may be involved. On the other hand, cell-substrate adhesion experiments and nanomechanical characterization using high-resolution atomic force microscopy showed that the stiffness (N=5, p>0.05), surface roughness (N=5, p>0.05) and adhesiveness (N=4, p>0.05) of MDA-MB-231 cells are not affected by stable α-ENaC overexpression.
Considering that α-ENaC is the pore-forming subunit necessary to form functional ENaC at the apical cell surface, this thesis next investigated the effects of inhibiting ENaC activity on breast cancer cell invasion and explored ENaC surface density in breast cancer cells. Surprisingly, ENaC inhibition with 5 µm amiloride (specific ENaC blocker) did not affect the suppression of MDA-MB-231 cell invasion induced by stable α-ENaC overexpression (N=3, p>0.05) but reduced the invasiveness of control cells (N=3, p<0.05). Additionally, the cell surface biotinylation assay revealed the absence of α-ENaC protein at the cell surface of MDA-MB-231 and wild-type MCF-7 cells, while stably overexpressing α-ENaC promoted cell surface α-ENaC level in MDA-MB-231 cells (N=3). Consistently, minimal channel activity was detected in wild-type MCF-7 cells (n=6) using a cell-attached patch clamp technique.
Taken together, the findings from this thesis describe novel effects of increased α-ENaC expression on TNBC cell invasion and morphology, likely involving regulation of F-actin dynamics. The work presented here also illuminates the potential influences of ENaC activity on breast cancer cell function. The findings of reduced TNBC cell invasion by α-ENaC overexpression further corroborate the notion that α-ENaC exerts a tumour-suppressive function in breast cancer, underscoring its potential as a molecular target for breast cancer therapies.